Shigellosis, also known as bacillary dysentery, is caused by several bacteria of the genus Shigella. Symptoms include diarrhea, abdominal pain, vomiting and fever. Generally, foodborne shigellosis involves a short incubation time, but symptoms can persist for up to 14 days. As few as 10 to 100 organisms have been shown to cause illness and secondary infections occur frequently. In the United States, Shigella accounts for up to 25% of the total cases of intestinal disease. The actual number of cases is estimated to be currently about 350,000. The proliferation of childcare facilities is playing an increasing important role in disease outbreaks.
Shigella infection is a serious public health problem in the United States and, as such, physicians are required to report cases to the Centers for Disease Control and Prevention. Further, recent changes in government regulations will make it incumbent upon the food industry to report enteric bacterial contamination of food. Public Health Laboratories, operated by the States, often must test specimens provided by physicians in order to identify the specific species of Shigella causing infection.
Shigella is an invasive pathogen that can be recovered from the bloody stool of an infected host. Invasive pathogens colonize the host's tissues as opposed to growing on tissue surfaces. Therefore, infections are also associated with mucosal ulceration, rectal bleeding, and drastic dehydration; fatality may be as high as 10-15% with some strains. Reiter's disease, reactive arthritis, and hemolytic uremic syndrome are possible sequelae that have been reported in the aftermath of shigellosis. Infants, the elderly, and the infirm are susceptible to the severest symptoms of disease, but all humans are susceptible to some degree. Shigellosis is a very common malady suffered by individuals with AIDS and AIDS-related complex, as well as non-AIDS homosexual men.
Transmission is frequently by ingestion of contaminated food or water. The organisms survive the gastric milieu and make it to the large intestine where the bacteria attach to specific host cells via invasins and then invade the epithelial cells lining the large intestines. Additional cell death is produced by elaboration of shiga toxins. Shiga toxin is a cytotoxin (or enterotoxin) that destroys epithelial cells lining the lumen of the large intestine; this causes bloody diarrhea characteristic of Shigella infection. Plasmid-mediated invasins determine the virulence of the particular strain of Shigella. Symptoms develop after an incubation period of 24 to 48 hours and include fever, abdominal pain and diarrhea.
The genus Shigella is a member of the family of Enterobacteriaceae and is thus related to Escherichia coli. Their DNA relatedness is very high, they are often difficult to differentiate biochemically, and they cross-react serologically. However, they have remained separate species for clinical reasons. Enterohemorrhagic E. coli (EHEC) is a defined subset of toxin-producing Shigella and at least one serotype (0157:H7) of EHEC can cause hemorrhagic colitis and hemolytic uremic syndrome, a potentially fatal complication.
The four species in the Shigella genus are sometimes referred to by a letter designation based on their serological antigen:                Serotype A—S. dysenteriae         Serotype B—S. flexneri         Serotype C—S. boydii         Serotype D—S. sonnei Serotype D is the causative agent of most cases of Shigella-related diarrhea. Although all Shigella species have been implicated in foodborne outbreaks at some time, S. sonnei is thought to be the leading cause of shigellosis from food. The other species are more closely associated with contaminated water. One in particular, S. flexneri, is now thought to be in large part sexually-transmitted.        
Current laboratory methods for detecting Shigella depend on growing the organism from a stool or food sample on culture plates and distinguishing it from other organisms on the basis of growth characteristics and biochemical tests. However, conventional testing currently is difficult. Growth on MacConkey agar may yield colonies of Shigella and E. coli that are both colorless, lactose negative, and about 2-3 mm in diameter. Growth of both organisms on sheep blood agar yields colonies that are alike, having a smooth appearance and 2-3 mm in diameter. Tube testing is widely used in reference and public health laboratories. Unfortunately, the media and tests used are not well standardized, and few laboratories use exactly the same procedures. Some 47 biochemical reactions are numerated to help distinguish between named species, biogroups, and enteric groups of the family Enterobacteriaceae. These tests are labor-intensive and costly. A genetic probe to a virulence plasmid has been developed by FDA and is currently under field test. Blood serology can also be used to distinguish one species from another. Although the Centers for Disease Control require distinguishing among the four major species of Shigella in its reporting statistics, it is difficult to do so.
Distinguishing between Shigella and E. coli is important for treating the infection. Diarrhea and other symptoms caused by infection with Shigella is one form of bacterial-caused intestinal inflammation in which antibiotic therapy has been clearly shown to reduce the duration and severity of symptoms and to shorten the period of fecal excretion of the organism. Thus, antibiotic therapy is generally recommended for all patients with Shigella diarrhea, except those with mild, self-limited disease. The recommended antibiotic is trimethoprim-sulfamethoxazole. Ampicillin-resistant strains are becoming a problem. There is no acquired immunity to infection and no vaccine exists. In contrast, there is no evidence that use of antimicrobial agents to treat E. coli 0157:H7 disease changes the course of the disease or is beneficial in any way. Distinguishing among the species of Shigella is not only important for proper reporting to the centers for Disease Control, but also for determining differing susceptibilities to antibiotics.
U.S. Pat. No. 4,724,205 relates to a composition for therapeutic treatment or diagnosis of the toxin associated with Shigella dysenteriae. U.S. Pat. No. 4,992,364 to Sansonetti et al. relates to a probe for DNA and a process for detection of Shigella and enteroinvasive strains of E. coli. The probe reportedly contained a nucleic acid sequence originating from the 140 mDa virulence plasmid of the M 90 T strain of Shigella flexneri, which permitted the in vitro diagnosis of syndromes of dysentery or diarrhea of the shigellosis type. The probe, however, could not distinguish either between E. coli and Shigella or between the different species of Shigella. A similar strategy was apparently employed by Lampel and Jagow (U.S. Pat. No. 5,041,372), but had the advantage of using synthetic oligodeoxyribonucleotides, which are more stable and easier and cheaper to make then the fragments prepared with restriction enzymes from the virulence plasmid. U.S. Pat. No. 5,084,565 to Parodos et al. relates to nucleic acid probes capable of specifically hybridizing to rRNA of E. coli and Shigella species and not to rRNA of non-E. coli/Shigella species. The hybridization assay could not distinguish either between Shigella and E. coli or between the different genera of Shigella. Faruque et al. (J. Clin. Microbiol. 30(11):2996, 1992) reports differentiation of Shigella flexneri strains by rRNA gene restriction patterns. Nastasi et al. (Res. Microbiol. 141:1163, 1990) reports the molecular analysis of strains of Shigella boydii isolated in northern and southern Italy.
Present detection methods using nucleic acid identity are not able to distinguish Shigella species from E. coli or Shigella species from each other. Therefore, known procedures are not completely satisfactory, and the present inventors provide herein compositions and methods for distinguishing Shigella from E. coli and species of Shigella from each other.